Proc. Natl. Acad. Sci. USAVol. 87, pp. 5888-5892, August 1990Biochemistry

Generation of collagenase-resistant collagen by site-directedmutagenesis of murine proal(I) collagen gene

(collagenase resistance/Mov]13/in vitro mutagenesis/imutant type I collagen)

HONG WUt, MICHAEL H. BYRNEt, ALEX STACEYt§, MARY B. GOLDRINGt, JAMES R. BIRKHEADt,RUDOLF JAENISCHt, AND STEPHEN M. KRANE*tWhitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; and tDepartment ofMedicine, Harvard Medical School and Medical Services (Arthritis Unit), Massachusetts General Hospital, Boston, MA 02114

Communicated by Jerome Gross, April 30, 1990 (received for review March 9, 1990)

ABSTRACT Collagenase (matrix metalloproteinase 1)cleaves type I, II, and III collagen helices at a specific sitebetween Gly-Ile or Gly-Leu bonds (residues 775 and 776,Pj-P1'). To understand the mechanism of collagen processing,mutations around the cleavage site have been introduced intothe cloned murine proal(I) collagen (Collal) gene. Thesemutant constructs have been transfected into homozygousMovl3 fibroblasts that do not express the endogenous Collalgene due to a retroviral insertion. Secreted triple-helical typeI collagens containing substitutions of Pro for Be (position 776)(P1') were not cleaved by human rheumatoid synovial colla-genase, whereas those containing substitutions of Met for BIe(position 776) were cleaved. Type I collagens containing doublesubstitutions ofPro for Gln-774 (P2) and Ala-777 (P2') were notcleaved regardless of whether they contained the wild-typeresidue Ile at position 776 or the substitution of Met for He atposition 776. The wild-type a2(I) chains derived from theendogenous Colla2 gene were also resistant to enzyme digestionwhen they were complexed with the mutant al(I) chains,indicating that the presence of normal al(I) sequences iscritical for cleavage of the a2(I) chains in the type I hetero-trimer.

a2(I) collagen chain (16). The introduction of in vitro muta-genized Collal genes into Movl3 cells is, therefore, a suit-able approach to evaluate the effect of specific Collalmutations on type I collagen synthesis and degradation.The collagenases cleave native type I, II, and III collagens

by hydrolyzing the peptide bond between residues Gly-Ile (orLeu) located at residues 775 and 776 of the helical portion ofthe al(I) chain to yield a larger three-quarter length fragment(TCA) and a smaller one-quarter length fragment (TCB) (1, 4,17). The region around the cleavage site is more hydrophobicthan other parts of the collagen molecule and deficient in thehydroxyproline and proline residues that stabilize the triplehelix. Our strategy was to generate mutations around thecollagenase cleavage site in a mouse Collal genomic clone,transfect the mutant gene into fibroblasts derived from ho-mozygous Movl3 mice, and then analyze the secreted col-lagen molecules for susceptibility to collagenase cleavage.These studies showed that type I collagen molecules con-taining wild-type a2(I) and mutant al(I) chains with aminoacid substitutions of Pro for Ile at position 776 or for Gln andAla at positions 774 and 777 were completely resistant toenzyme digestion.

Collagenases are the only enzymes characterized so far thatare capable of degrading undenatured type I, II, and IIIcollagens extracellularly at neutral pH (1-5). A genetic ap-proach to study the role of collagenases in physiological andpathological processes of connective tissue remodeling hasbeen hampered by the lack of existing mutations in thecollagenase genes. An alternative approach would be tointroduce mutations into the collagen molecule that wouldrender it resistant to cleavage by collagenase. Many inheritedor spontaneous mutations in humans that lead to alterationsin the structure and function of collagen have been described(6-11), but none has been reported that results in alteredsusceptibility to collagenase cleavage.We have used the Movl3 mutant to analyze sequence

requirements for collagenase cleavage of the proal(I) colla-gen chain by introducing amino acid substitutions into theCollal gene. In the Movl3 mouse strain, the Collal gene isinactivated due to a proviral insertion while transcription ofthe Colla2 gene is not affected (12-14). Fibroblast cell linesderived from Movl3 homozygous embryos are deficient notonly in the production of al(I) chains but also in the produc-tion of stable a2(I) collagen chains because triple helices arenot formed in the absence of proal(I) chains (15). Transfec-tion of human or mouse Collal genes into homozygousMovl3 cells, however, results in the synthesis and secretionof triple-helical type I collagen containing the endogenous

MATERIALS AND METHODSConstruction of Mutant Collal Collagen Gene. All con-

structs were prepared as described by Stacey et al. (18) usingderivatives of the murine Collal genomic clone 10D (16, 37).The nucleotide and amino acid sequences ofthe wild type andeach mutation are given in Fig. 1. To facilitate mutagenesisin or around the collagenase cleavage, a Kpn I-Sac II"cassette" was constructed in which the Kpn I site atposition 2026 (within an intron) was destroyed, rendering theother Kpn I site (at position 1809) unique, and amino acidsVal-Val (782 and 783) were replaced by Ala-Ala to create anew Sac II site (mutants II-V).

S1 Nuclease Protection Assay. Mutant constructs weretransfected along with the selective markers, pAG60 andpSV2-neo, which contain the neomycin phosphotransferasegene conferring resistance to the drug G418 into the Movl3cells or NIH 3T3 cells as described (19, 20) at a molar ratioof 7:1. NIH 3T3 cells were selected in G418 at 1 mg/ml(GIBCO) and Movl3 cells were selected in G418 at 0.3 mg/mlbecause they had different sensitivities for optimal selection.

Total RNA from individual clones was prepared accordingto the method of Auffray and Rougeon (21). S1 nucleaseanalysis was performed to test the presence of mutanttranscripts as well as the a2(I) collagen transcripts by estab-lished procedures (18, 22, 23). Five micrograms ofRNA fromeach clone was ethanol precipitated with a mixture of the S1probes that were empirically determined to be in excess by

§Present address: Department of Physiology, Colorado State Uni-versity, Fort Collins, CO 80523.

5888

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 87 (1990) 5889

using increasing amounts of RNA. After denaturation at80'C, the hybridization was carried out at 50'C for 16-20 hr.

Collagenase Digestion. Preparation of labeled procollagenwas performed as described (24). Samples were dissolved incollagenase buffer (0.15 M NaCl/0.05 M Tris HCl, pH 7.4/0.005 M CaCl2/0.25 M glucose) and incubated at 20'C withtrypsin-activated procollagenase for various periods up to 18hr. Procollagenase was obtained from medium conditionedby primary cultures of rheumatoid synovial fibroblasts, pre-cipitated with 50% saturated (NH4)2SO4, and chromato-graphed on columns of Ultrogel AcA-54 (LKB, Bromm,Sweden) (25). The peak containing procollagenase, whichwas assayed after activation with trypsin (TRPCK; CooperBiomedical), was collected and used. After incubation withcollagenase, the collagen samples were mixed with loadingbuffer with or without 0.1% 2-mercaptoethanol and fraction-ated by electrophoresis on 7% polyacrylamide SDS/PAGEgels (24). Fluorography was performed as described (24).

Determination of Denaturation Temperature of Collagens.Denaturation (melting) temperature was determined by mea-suring the susceptibility of mutant collagens to trypsin diges-tion over a range of temperatures by a modification of themethod of Constantinou et al. (26).Cyanogen Bromide (CNBr) Digestion. Labeled type I col-

lagen chains or collagenase cleavage products ofthese chainswere first separated by SDS/PAGE as described. To identifybands in wet gels, proteins were first fluoresceinated (27).Following SDS/PAGE, bands identified under UV light werecut out of the wet gels, solubilized using an electroeluter(model 422, Bio-Rad) according to the manufacturer's in-structions, and freeze-dried. For CNBr cleavage sampleswere then redissolved in CNBr in 70%o (vol/vol) formic acidand maintained at 15-20°C for 3 hr. The CNBr/formic acidwas then removed by freeze-drying. In other experiments,appropriate bands were identified in the slab gels, excised,incubated directly in 5% CNBr in 70% formic acid, andincubated for 2 hr at 15-20°C followed by water washesaccording to the procedure of Byers et al. (9). The resultingpeptides were separated by SDS/PAGE in 10%o or 12%acrylamide or 10-20% gradient gels (Integrated SeparationSystems, Newton, MA) and analyzed by fluorography.

RESULTSMutagenesis of Collal Gene and Derivation of Cell Clones

Producing Mutant mRNAs. Five different mutations wereintroduced into a murine Collal genomic clone that includesabout 17 kilobases (kb) of coding region, 3.7 kb of 5' and 3 kbof 3' flanking sequences, and a 21-base-pair (bp) polylinkerfragment inserted into the Xba I site in the 5' untranslated

Kpn I Sau l 4

region (16, 18). The mutant constructs encoded variousamino acid substitutions around the collagenase cleavage sitespanning amino acid residues between positions 774 and 783(Fig. 1). The presence of mutant proal(I) and wild-typeproa2(I) collagen transcripts was determined by S1 nucleaseprotection analysis. As expected, the 196-base fragmentprotected by the mutant transcript was detected in trans-fected Movl3 cells (Fig. 2A), whereas the 196-base and the95-base protected fragments were seen in transfected NIH3T3 clones (Fig. 2B). No 95-base protected fragment wasseen in transfected Movl3 cells (not shown in this particulargel), as shown previously (18). The 122-base fragment char-acteristic of the wild-type proa2(I) collagen transcripts wasdetected in all clones. The relative levels of these protectedfragments differed among clones, indicating that differentamounts of proal(I) collagen transcripts were produced fromthe mutant constructs and that the ratio of al(I):a2(I) variedfrom clone to clone.Type I Collagen Containing Mutant al(l) Chains Is Resis-

tant to Collagenase Cleavage. Type I collagen containingmutant al(I) chains was prepared by limited digestion ofproteins with pepsin after labeling the cells with [3H]proline.Whereas untransfected Movl3 cells did not produce detect-able type I collagen, in agreement with previous reports (13,15, 16) (Fig. 3A, lane 3), Movl3 cells transfected with themutant constructs produced type I collagen consisting of themutant al(I) chains and the wild-type a2(I) chains (Fig. 3A,lanes 5, 7, and 9). Different amounts of mutant al(I) chainswere produced by individual transfected clones, and theamount of type I collagen as well as the al(I):a2(I) ratiovaried from clone to clone (Fig. 3A), consistent with thevariations in collagen transcriptional levels.To test the susceptibility of mutant type I collagen to

collagenase digestion, the labeled collagens were exposed topartially purified rheumatoid synovial fibroblast collagenase.An excess of unlabeled rat tail collagen was added as carrier.Digestion conditions (18-24 hr at 20°C) were chosen to allowcomplete cleavage of the carrier collagen (Fig. 3B) as well aslabeled type I and III collagens synthesized by control NIH3T3 cells (Fig. 3A), indicating that the enzyme was in excess.As shown in Fig. 3A, lanes 1, 2, 5, and 6, wild-type type I andtype III collagens were completely digested by collagenase, asindicated by the presence of the expected cleavage productsTCAal(I), TCBal(I), TCAa2(I), TCBa2(I), TCAal(III), and TCBal(III).Although the TCAal(l) and TCBal(III) migrated similarly undernonreducing conditions (lanes 2 and 6), both cleavage productswere well separated under reducing conditions (data notshown). The type V collagen chains were not cleaved bycollagenase under this condition, consistent with previousobservations (29). In contrast to wild-type collagen produced

CollagenaseHpa It CleavageHpI1

GGT ACC CCT GGA CCT CAG GGT ATT GCT GGA CAA CGT GGT GTG GTC GGT CTT CCC GGT +GLY TIR PRO GLY PRO GLN GLY I LE ALA GLY GLN ARG GLY VAL VAL GLY LEU PRO GLY

774 775 776 777 782 783

p3 p2 p1 p1l p2' P3' P4' pi' p 4 p7' putant Pst I

I CCTGCA GGA.....0.PRO ALA GLY I

Sac 11

II ... CCTCCG GGT ATT CCT...... * -GCCGCGGGT -PRO PRO GLY ILE PRO ALA ALA GLY

Sph II

... ... ... ... CCT CCG GGC ATG CCT ... ... ...

PRO PRO GLY MET PRO

Nia HIl... ... ... ... CCT CAG GGC ATG GCT ... ... ...

PRO GLN GLY MET ALA

... GCC GCG GGT ...ALA ALA GLY

..... S

... GCC GCG GGT ... ... ...

ALA ALA GLY

... GCC GCG GGT ..- SALA ALA GLY

Fli. 1Pn. -nr-- ndinronttLrIU. 1. at;UqUnU~CS UUJULt;CHL LU

O collagenase cleavage site (arrow)in wild-type murine Collal geneand in mutant constructs de-

low scribed in this report. The resultsof collagenase cleavage are shownon the right after each construct.

O +, Sensitive to collagenase diges-tion; 0, resistant to collagenasedigestion; slow, 31-39o of type I

low collagen is cleaved under the con-ditions described in the text.

Ml

III

IV

V

Biochemistry: Wu et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

A MAo 13

1 2 3 4 5

B 3T3

bases1 2 3

ms am -196

Abaser

-_W _ -- 1 96

um -- 122 - - 1 22

_-- 95

_

m_m_

CollalC

he0 Exon I NA- Intron lProtected Fragments:

Exogenous mRNA 57 196 bases

EndogenousmRNA 95 bases

Probe32P v B

FIG. 2. S1 nuclease analysis of the expression of wild-type(endogenous) and mutant (exogenous) Collal genes in Movl3 andNIH 3T3 (3T3) cells. Five micrograms of total RNA from eachtransfected clone was used. Hybridization was carried out at 50TCwith a mixture of mouse al(I) (depicted in C) and a2(I) S1 probes.The a2(1) probe, derived from pAZ1002 (28), functions as an internalcontrol by giving rise to a 122-base protected fragment. (A) Mutantgene expression in Movl3 cells. Lane 1, untransfected Movl3 cells;lanes 2-5, Movl3 cells transfected with mutants II, I, IV, and V,respectively. (B) mRNA from mutant IV transfected NIH 3T3 cells.Lanes 1-3, mRNA extracted from clones 4, 8, and 11, respectively.(C) Scheme of the S1 nuclease analysis used to distinguish endog-enous and exogenous al(I) transcripts. The probe was 600 bases inlength and included the 21-base insert as shown (triangle). Theexogenous transcripts protect a 196-base fragment, whereas theendogenous transcripts protect a 95-base fragment.

by NIH 3T3 cells (Fig. 3A, lane 2), the al(I) collagen chainsproduced by Movl3 cells transfected with mutants I and II (seeFig. 1) were completely resistant to collagenase digestion (Fig.3 A and B). Importantly, the wild-type a2(I) chains in theheterotrimeric collagen molecules were not cleaved by theenzyme, suggesting that the correct sequences of both al(I)and a2(I) chains in register are required for the enzymeactivity. Under the same conditions, -31-39% of type Icollagens produced by Movl3 cells transfected with mutantsIII and V were cleaved by collagenase (data not shown). Thesefindings suggest that the enzyme recognizes sequences beyondthe highly conserved region defined previously (17).To examine whether the secreted mutant collagen was in

the native helical conformation, the melting temperatures ofthe mutant as well as wild-type collagens were compared bydigestion with trypsin at different temperatures (see Materi-als and Methods). All mutant type I collagens tested had amelting temperature of -380C, indistinguishable from that ofthe wild-type collagen (data not shown).Mixed Collagen Type I Heterotrimers Containing Mutant

and Wild-Type al(I) Chains Are Sensitive to CollagenaseCleavage. The results described above indicated that hetero-trimers consisting of two mutant al(I) chains and one wild-type a2(I) chain were resistant to cleavage by collagenase.The following experiments were designed to examinewhether a single mutant al(I) chain in the triple helix wouldbe sufficient to render the heterotrimer resistant to collagen-ase cleavage.Mutants IV and V carrying a Met at position 776 had been

engineered from mutants II and III. Since Met and Ile havesimilar hydrophobicity, we expected that mutants IV and Vwould behave similarly to their parental constructs. This was

;oiwIager .- c r erMutant Cov c e:

Ui

-I I-1a-x2, I: --- _o

-

*1'11,_

.,Is

); w'ag erAMulta

vrer;>. Ad Iae

4-

_ _ IYZ

iA cX iZ

,r ALuv ;'1

A.lC"l<-V.-C

Co Irgeige asL

FIG. 3. Cleavage of wild-type (wt) and mutant collagens bysynovial fibroblast collagenase. (A) L-[5-3H]Proline-labeled colla-gens were isolated from medium proteins by limited pepsin digestionat 00C. Collagenase digestion was carried out at 20'C for 18 hr withsynovial fibroblast collagenase that is sufficient to digest 1.7 ,g ofcollagen per hr. Under these conditions, the labeled collagen (typesI and III) from the NIH 3T3 (3T3) cells (lane 2) as well as 10 Ag ofunlabeled carrier rat tail tendon collagen (see B) were digestedcompletely, yielding the characteristic reaction products. Onlynonreducing conditions of SDS/PAGE are shown. The faint bandseen in lane 4 just below that of TCA1(III) is the disulfide-linkedTC010(tI1) trimer. The band that migrated similarly to TCAal(I) seenin lanes 3, 5, 7, and 9 is a partial pepsin digestion product ofcollagensother than type I. Fluorograms were exposed for 30 days (lanes 1 and2), 11 days (lanes 3 and 4), 1 day (lanes 5 and 6), 3 days (lanes 7 and8), and 4 days (lanes 9 and 10). (B) Collagenase cleavage of labeledmutant II collagen and unlabeled carrier rat tail tendon collagen.Labeled collagen derived from mutant II was incubated with colla-genase under the conditions described above. Five-sixths of thesample was analyzed by SDS/PAGE and fluorography (lanes 1 and3). Fluorograms were exposed for 4 days. Proteins in the remainingone-sixth ofthe sample were also resolved by SDS/PAGE, and silverstaining was used to detect the unlabeled carrier rat tail tendoncollagen (lanes 2 and 4). Only nonreducing conditions are shown.Note that the carrier al(I) and a2(I) collagen chains were digestedcompletely. The faint band remaining in lane 4 was a2(V). Themutant collagen was not digested under the same conditions (lane 3).

shown to be the case in experiments where type I collagenswere isolated from Movl3 cells transfected with mutants IVand V and subjected to collagenase cleavage (data notshown). Met at position 776 introduces a site for CNBrcleavage and therefore allows the wild-type al(I) chain to bedistinguished from the mutant al(I) chain in a triple-helicalmolecule. In wild-type al(I) chains, CNBr cleavage gener-ates fragment CB7; in contrast, al(I) chains derived frommutants IV and V will be cleaved at position 776, generating

_

-b

-~~~4

5890 Biochemistry: Wu et al.

dOftb

;.D

Proc. Natl. Acad. Sci. USA 87 (1990) 5891

A o (1)

8 3,t7 16

CNBrCollogenase

peptide d (I) TCAaI(l) TCAOMI(Di TCB o 1

7 --

83+7a 7b

8-7a--6 -- oc(i) (Mutants IVYV)

3 __ 7o 7bt8 3+-3-f--%;

3T3 3T3 3T3 Mov 13B Transfected Tronsfected Transfected Transfected

Mutant 117 Mutant IV Mutant IV Mutant 1VCN[3r alone 4 Clone 8 Clone l1peptide 1 2 3 4

6 .- _1

Mutant UVin

C 3T3 Mov 3

1 2 3 4

a

Mutant TV in3T3

Clone Clone Clone4 8 11

5 6 7 8 9 10

EM *_ -oa2(I?a mem-Z *o -

*0 Qa40 -TCAC2fl)

fragments CB7a* and CB7b* (Fig. 4A). The migration posi-tion of the larger fragment, CB7a*, in the gel can be deter-mined by digesting the wild-type collagenase cleavage prod-uct, TCAaL(I), with CNBr. This produces a CB7a fragmentthat should have electrophoretic mobility identical with thatof CB7a* (see Fig. 4A).The mutant constructs were transfected into NIH 3T3

cells, and mutant as well as the wild-type al(I) procollagentranscripts were quantified by the S1 nuclease protectionanalysis (Fig. 2B). Collagen was collected from three differ-ent mutant IV transfected clones. The relative amounts ofCB7a* present in al(I) chains before incubation with colla-genase were quantified by optical densitometry ofPAGE gelsand expressed relative to CB6, as shown in Table 1. Fig. 4Bshows that fragment CB7a* was prevalent (about 80o oftotalby densitometry) in type I collagen secreted by clone 4,indicating a high abundance of mutant al(I) collagen chainsin heterotrimers or al(I) homotrimers. In contrast, the al(I)chains secreted by clone 8 contained CB7, characteristic ofwild-type al(I) chains, and barely detectable CB7a*. Mutantal(I) chain comprised about 61% of the total al(I) chainproduced by clone 11. Correction for the distribution oflabeled proline residues was made by normalizing to theratios determined in Movl3 cells transfected with mutant IV.The extent of degradation of type I collagen by collagenase

was quantified (Table 1) by densitometry of the fluorogramsshown in Fig. 4C. Since approximately two-thirds of the typeI collagen produced by clone 11 was cleaved by collagenase,yet only 39% of the al(I) chains were wild type, some of thecleaved heterotrimers must have contained mutant al(I)chains. It seems, therefore, that a single mutant al(I) chainin the triple helix is not sufficient to render the heterotrimerresistant to collagenase cleavage.

-TC Bo.Ul 11

ws -S~~~~~~~C8*2llDCollagenase 0 + o + 0 + 0 +

FIG. 4. CNBr digestion of labeled wild-type and mutant al(I)chains. (A) Identification of CNBr cleavage products. The al(I)chains synthesized by NIH 3T3 cells as well as its collagenasecleavage product, TCAa (I), were digested by CNBr and resolved in10%o acrylamide gels. In the scheme on the right, the positions ofMetresidues in the al(I) chain are indicated by the vertical bars; thenumbering of the CNBr peptides (30) is shown. Since collagenasecleaves within peptide CB7, the product, TCAal(l), would contain ashorter CB7 peptide, which we designate CB7a. In mutants IV andV, which contain an additional Met residue at position 776, CNBrdigestion of the al(I) chains would produce a fragment identical withCB7a, which we designate CB7a*. The migration position of CB7a(or CB7a*) is shown in the digestion products of TCAal(I). Fluoro-grams were exposed for 6 days. (B) CNBr cleavage products frommutant IV transfected Movl3 and NIH 3T3 cells analyzed bySDS/PAGE and fluorography. The al(I) chains derived from threetransfected NIH 3T3 (3T3) clones and one transfected Movl3 clonewere electroeluted from the gel and digested with CNBr. Fluoro-grams were exposed for 30 days. For clone 8 from 3T3 cellstransfected with mutant IV, no CB7a* was detected, as shown here(lane 2), or only a faint band was detected in other preparationsexposed for longer periods. (C) Collagenase cleavage of wild-typeand mutant IV collagens. Labeled collagens from NIH 3T3 cells,transfected Movl3 and NIH 3T3 cells (with the mutant IV construct)were incubated with collagenase. Note that type I collagen from 3T3cells was digested completely by collagenase (lane 2). Mutant IVcollagen, the only type I collagen secreted by Movl3 cells (lane 4),was not digested. Collagen from clone 8 was also completelydigested, whereas that of clones 4 and 11 was partially resistant. Thewild-type a2 (I) chains in the mutant type I collagen trimers (lanes 4)were not cleaved by the collagenase. Fluorograms were exposed for7 days (lanes 1-4) or 1 day (lanes 5-10).

DISCUSSIONThe triple-helical region of interstitial collagens is highlyresistant to all proteinases except collagenase. To study thefunction of collagenase in the processes of connective tissueremodeling, we have produced five mutations in the Collalgene that encode alterations of amino acids around thecollagenase cleavage site in the al(I) chain. The mutant geneswere expressed in Movl3 cells, and secreted type I collagenwas assayed for susceptibility to collagenase cleavage. Allmutations affected the efficiency of enzyme cleavage.Whereas the substitutions at positions 782 and 783 (present inmutants II-V, see Fig. 1) resulted in a slower rate ofcleavage,a substitution at position 776 and at positions 774 and 777resulted in complete resistance to enzyme attack. Althoughvaluable information had been provided by studies of thesusceptibility of small synthetic peptides with higher Km tocollagenase (17), the relative rates of hydrolysis of thesynthetic peptides could not, in themselves, explain whycollagenase cleaves its natural substrates only at the specificGly-Ile or Gly-Leu bonds and cleaves denatured collagens atrates slower than those of native collagens. Our resultsprovide evidence that collagenase requires specific aminoacid sequences to cleave native helical type I collagen.

Table 1. Susceptibility of wild-type and mutant collagensto collagenase

Wild-type Mutant IV CollagenaseClone al(I), % al(I), % digestion, %

4 21 79 308 -100 =0 >94

11 39 61 67

Calculations were made from densitometry of fluorograms similarto those in Fig. 4 B and C. See text for details.

Biochemistry: Wu et A

Proc. Natl. Acad. Sci. USA 87 (1990)

Ile-776 -- Pro-776. Collagenase cleaves all interstitial col-lagens between the P1-Pj' of Gly-Ile or Gly-Leu (31). Ile andLeu have a similar degree of hydrophobicity. In contrast, Proresidues are more accessible to solvent (32). The substitutionof Ile -- Pro in mutant I, therefore, was expected to changethe hydrophobicity of the cleavage domain. The mutantprotein was completely resistant to enzyme digestion, sug-gesting that the presence of a hydrophobic residue at thecleavage site is essential for enzyme action. Consistent withthis hypothesis is the observation that substitution of Ile-776-- Met, which does not result in appreciable change inhydrophobicity at the cleavage site, had no effect on the rateof cleavage (Fig. 1, compare mutants III and V).

Gln-774 -- Pro, Ala-777 Pro. The consensus sequenceat the cleavage site is found >27 times in type I, II, III, andIV collagens from different species, suggesting that se-quences flanking the cleavage site are important for enzymebinding or catalytic action. To understand the function ofsurrounding sequences that underlie specificity at the cleav-age site, we generated mutants II and IV. In these mutants,Pro was substituted in the -Yaa- position of the Gly-Xaa-Yaa-triplet. Such substitutions are predicted to stabilize the triplehelix (31, 33), particularly because Pro residues in the -Yaa-position are substrates for prolyl 4-hydroxylase (34). Al-though we were unable to detect a change in denaturationtemperature of the native mutant collagen, the Pro substitu-tions rendered the protein completely resistant to collagenasedigestion. These results are consistent with the hypothesisthat local helix instability may be a factor that determines theability of collagenase to cleave native collagens.

Val-782,783 -* Ala. These substitutions were introducedinto the gene in order to generate a cassette that wouldfacilitate the introduction of oligonucleotides containing thevarious point mutations into the Collal gene. Contrary to ourexpectations, however, the rate of collagenase cleavage oftype I collagens containing mutation III and V was slowerthan that of the wild-type collagen. The P7'-P8' sites incollagens I, II, and III contain one or two hydrophobicresidues such as Val, Ile, Leu, or Phe. Because Ala residuesare more hydrophilic than Val, and substitutions of Val-782,783 Ala are not conservative, our results suggest thathydrophobicity in this domain may be important for efficientcleavage.Our results also suggest that cleavage of the a2(I) collagen

chain in the type I heterotrimer is dependent on the presenceof cleavable sequences in the al(I) chains. The data de-scribed in Fig. 4 suggest, however, that a single mutant al(I)chain in a mixed heterotrimer does not prevent cleavage of allthree chains of the triple helix. Nevertheless, resistance of asingle mutant chain would probably prevent dissociation ofpartially digested wild-type chains in the heterotrimer.We cannot predict what effect the expression of the mutant

genes might have on development when introduced intotransgenic animals. It is possible that type I collagen that isresistant to cleavage by collagenase would interfere withtissue remodeling during early morphogenesis. We do notknow, however, whether a collagenase-resistant Collal genewould act in a dominant negative manner (18, 35, 36),resulting in disturbance of embryonic development of awild-type embryo carrying a mutant transgene. Alterna-tively, the collagenase-resistant Collal gene may cause arecessive phenotype. This could be analyzed in vivo bycrossing a transgenic mouse carrying the mutant gene withMovl3 mice, thus eliminating the wild-type Collal genes andrevealing the phenotype of the recessive mutation. If thedevelopmental consequences of collagenase-resistant Collalgenes are not severe and live mice can be obtained, theyshould provide useful models for human inflammatory dis-eases in which remodeling of collagen in connective tissuesis disturbed.

This work was supported by National Institutes of Health Grants5R35 CA44339 and P01 HL41484 to R.J. and National Institutes ofHealth Grants AR03564 and AR07258 to S.M.K. This study wasperformed as part of the Ph.D. thesis requirement of H.W. at theDepartment of Biological Chemistry and Molecular Pharmacology,Harvard Medical School.

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Schwartz, R. C. (1988) Am. J. Hum. Genet. 42, 237-248.10. Cetta, G., Ramirez, F. & Tsipouras, P. (1988) Ann. N. Y. Acad.

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